Dopamine regulates water and electrolyte excretion by actions on renal hemodynamics and tubules and by modulation of the action of other vasoactive and renoactive hormones. Direct actions are exerted at several nephron segments (proximal tubule, thick ascending limb of Henle. cortical collecting duct), via several transporters, and are mediated by multiple mechanisms. D1 receptor function is negatively regulated by a G protein-coupled receptor kinase, GRK4gamma that has been thought to have minimal function or even nonfunctional. Thus, inhibition of transcription/translation of GRK4 enhances the ability of D1 receptors to stimulate adenylyl cyclase activity Expression studies in Chinese hamster ovary cells show that the gene variants of GRK4y constitutively impair D1 receptor function. Furthermore, hyperexpression of the gene variant GRK4gamma A142V in mice impairs renal D1 receptor-mediated natriuresis and produces hypertension. The participation of GRKs via clathrin, in the desensitization of G protein-coupled receptors (including the D1 receptor) is well established. However, preliminary data show that the initial stages of D1 receptor desensitization are independent of URK. This GRK-independent regulator of D1 receptor function may involve caveolin-2. The overall hypothesis is that in renal proximal tubules, D1 receptor function is regulated by GRK-dependent, clathrin-regulated, and GRK independent, caveolin-2-regulated pathways. Specific aim 1 will test the hypothesis that GRK4, specifically, the isoform GRK4gamma, is the major GRK that specifically regulates D1 receptor function. Specific aim 2 will test the hypothesis that in renal proximal tubules, D1 receptors exist in two microdomains. The D1 receptor/caveolin-2 pool is responsible for the initial stages of desensitization while the D1 receptors/clathrin pool is responsible for the later stages of desensitization or down-regulation and resensitization. Using morphological, molecular biological, biochemical, and physiological end points, we will determine the interactions among D1 receptors, caveolin-2, GRK4, clathrin, and signaling proteins in rat renal proximal tubule cells. The understanding of the pathways of the regulation of D1 receptors in the kidney may lead to a better understanding of the dysregulation of D1 receptors that exists in disease states. Indeed, a constitutively hyperphosphorylated and desensitized D1 receptor caused by activated GRK4gamma gene variants may be a cause of a subset of human essential hypertension.